Fecal pollution in water is associated with several thousand of human mortalities per day, serving as source of pathogen transmission.
A myriad of indicators are known in the art for monitoring fecal pollution, herein after also referred as “faecal contamination”. In addition to classical bacterial indicators, several groups of bacteriophages (also called simply “phagues”) infecting enteric bacteria have been suggested for determinating the fecal pollution level in water. Bacteriophages are viruses that infect and replicate within a bacterium. When compared to classical bacterial indicators for detection of faecal contamination, bacteriphages are more resistant to disinfection and other inactivation processes and diffuse further distances from pollution sources. Therefore, bacteriophages may serve as a better predictor of water and food quality. The potential value of bacteriophages as quality indicators in water and food has been heavily investigated and reviewed (Lucena F.; Jofre J. “Potential use of bacteriophages as indicators of water quality and wastewater treatment processes.” In SABOUR, P. M.; GRIFFITHS, M. W. (ed). Bacteriophages in the Control of Food- and Waterborne Pathogens. ASM Press, Washington D.C., 2010, pag 103-118). Among bacteriophages, somatic coliphages, which are phages able to infect and replicate in E. coli and some bacterial species freom faecal origin closely related to E. coli, have recently been included in water quality guidelines, such as those for ground water in US (USEPA, 2006, National Primary Drinking Water Regulations: Ground Water Rule; Final Rule; 40 CFR Parts 9, 141 and 142. Federal Register, vol. 71, No. 216. p. 65574-65660. Environmental Protection Agency. Washington D.C.) or for water recycling in the State of Queensland in Australia (Quensland Government, 2005, Water recycling guidelines. Queensland State EPA, Brisbane. Australia).
Several strains of E. coli and assay media yield different results in the determination of somatic coliphages. In order to avoid this source of variability in the determination, standarized methods for detecting and quantifying this group of phages have been established and are available at present time (ISO 10705-2. 2000; APHA, AWWA and WPCF, 2001; or USEPA 2001a. Method 1602). All these standarized methods use E. coli C as host strain: either the wild strain ATCC13706 (APHA, 2001) or its nalidixic resistant mutants WG5 (ISO 10705-2, 2000) and CN13 (USEPA 2001a).
The APHA standard method has been however reported to perform poorly, while methods based on host strains WG5 and CN13 have shown better accuracy in water environments. Available data indicate that these E. coli strains are susceptible to the same bacteriophages (Muniesa et al., 2003, “Bacterial host strains that support replication of somatic coliphages”. Antonie van Leeuwenhoek 83: 305-315) and, when used in the detection of somatic coliphages, they both provide similar results (Guzman et al., 2008, “Evaluation of Escherichia coli host strain CB390 for simultaneous detection of somatic and F-specific coliphages”. Appl. Environ. Microbiol.; 74(2):531-4).
The current ISO protocol using E. coli WG5 for detection of somatic coliphages is a multiple-step procedure that involves coliphage replication in exponential-growth-phase cells of the host E. coli followed by a spotting on seeded agar for plaque confirmation. The method is laborious and time consuming, results not being available until at least 18 hours. Additionally, despite its good performance, the lack of a ready-to-use test limits the implementation of this protocol in many laboratories for general water and food management policies.
WO 9428179 A1 reports an alternative method (“Fast phage”) for detecting somatic coliphages based on the measurement of phage mediated release of intracellular β-galactosidase. The method comprises incubating a coliphage-containing water sample in a medium than comprises an E. coli host that is susceptible to infection by coliphages. The incubation medium also contains a compound that induces the expression of high levels of intracellular beta-galactosidase in the E. coli host. The mixture is then incubated with a labelled substrate for beta-galactosidase, which will undergo a detectable change when cleaved by this enzyme. When the sample contains coliphages, phage-mediated lysis of the E. coli host releases the intracellular beta-galactosidase enzyme, which in turn cleaves the labelled substrate enabling visual detection. The method shows the great drawback of low sensitivity. In addition to requiring concentration of the sample, for detection of low phage titers the document discloses long incubation times with the labelled substrate. However, incubations exceding two hours are discouraged (possibly because longer incubations would yield high number of false positives). It follows that the method's sensitivity and specificity are unbalanced. Detecting low titers (high sensitivity) is jeopardized by false positive results (low specificity), while performing the method with high specificity inevitably leads to a bad sensitivity.
Salter introduced the “Fast Phage” concept in the standard coliphage somatic determination (EPA Method 1601) (Salter et al., 2010, “Proposed modifications of Environmental Protection Agency Method 1601 for detection of coliphages in drinking water, with same-day fluorescence-based detection and evaluation by the performance-based measurement system and alternative test protocol validation approaches”; Appl Environ Microbiol. 76(23):7803-10). The modification of the EPA method 1601 including Fast phage intended to detect low phage titers with the same performance as the standard method. Thereto, the method requires an enrichment step consisting of incubating the phage-containing sample with the E. coli host and beta-galactosidase inducer for at least 5 h. This step is necessary in order to increase the amount of phages in the sample. The enriched culture is then incubated with a flourescence-labelled beta-galactosidase substrate for further 3 h. The whole process implies at least 8 hours for final detection. As reported by the authors, false positive results are also an issue when peforming this method.
A different approach has been described by Guzmán (Guzman et al., 2009, “Detection of somatic coliphages through a bioluminescence assay measuring phage mediated release of adenylate kinase and adenosine 5′-triphosphate”; J Virol Methods. 161(1):107-13). The disclosed method detects somatic coliphages after phage infection of E. coli WG5 and lysis-mediated release of the bacterial host's adenylate kinase (AK) and adenosine 5′-triphosphate (ATP) by detection of a bioluminescent signal. This approach requires at least 3 hours for incubation of the E. coli host with the phage-containing sample plus the time needed to extract the AK before proceeding with the detection. Expensive laboratory equipment and reagents are required for extraction and detection of AK so that this method suffers from considerable drawbacks related to high costs and impossibility of point-of-use implementation.
In view of all the above, there is still a need to provide fast, cost-effective tests for the detection of somatic coliphages with high specificity and sensitivity.